The apicomplexan parasite Cryptosporidium is a significant cause of diarrheal disease worldwide, particularly in immunocompromised hosts such as AIDS patients. The overall goal of this project is to investigate the role of subtilisin-like serine proteases (subtilases) of C. parvum in host-parasite interactions. Two genes encoding putative subtilases (designated CpSUB1 and CpSUB2) have been identified in the C. parvum genome but have not yet been investigated. Preliminary studies show that both genes are expressed during C. parvum infection in vitro suggesting that the proteins they encode are likely to be important in host-parasite interactions. The hypothesis is that CpSUB1 and/or CpSUB2 process surface and apical complex proteins such as gp40/15 and mediate C. parvum infection in vitro. In the first specific aim we will quantify mRNA expression of CpSUB1 and 2, identify the precursor, processed and mature forms of the CpSUB1 and 2 proteins expressed in C. parvum, investigate their post-translational processing and determine their subcellular localization. In addition, we will express enzymatically active recombinant CpSUB1 and CpSUB2 and characterize their enzymatic activity. In the second specific aim we will determine whether either subtilase processes recombinant gp40/15. In addition we will determine whether propeptide inhibitors or antibodies to the CpSUBs inhibit C. parvum infection of intestinal epithelial cells in vitro. The long term goal is the rational, structure-based design of inhibitors of these enzymes as drugs to prevent or treat cryptosporidiosis. Project Narrative/Relevance The intestinal parasite Cryptosporidium is a significant cause of diarrheal disease worldwide, particularly in immunocompromised hosts such as AIDS patients. In this project we will study enzymes that are important for infection of host cells by the parasite, with the long term goal of developing inhibitors of these enzymes as drugs for cryptosporidiosis. Since there is no consistently effective treatment available for this disease, these studies are important for development of new drugs.